Recombinant gelatin-like proteins for use as plasma expanders

ABSTRACT

The invention relates to compositions suitable for plasma substitution comprising as a plasma expander a recombinant gelatin-like protein. Characteristic is that the gelatin-like protein essentially is free of hydroxyproline. This absence of hydroxyproline prevents the composition from gelling and thus allows the use of high-molecular weight proteins in order to establish a suitable colloid osmotic pressure. Specific advantage of the gelatin-like proteins is that these avoid the risk of anaphylactic shock that exists in conjunction with the use of commercially available preparations.

FIELD OF THE INVENTION

The invention relates to the use of recombinant gelatin-like proteins—orpolypeptides—as plasma expanders and to compositions suitable for plasmasubstitution comprising such a plasma expander.

BACKGROUND OF THE INVENTION

A well established application of gelatin is the use as a colloid insolutions as substitutes for plasma. Such plasma substitutes can be usedfor controlling circulating blood volume in the management of shockresulting from for instance hemorrhages or burns. Care should be takenthat the gelatin solution is made sterile, pyrogen and antigen free, andas the result of the average molecular size, is capable of maintaining adesired colloid osmotic pressure. In order to maintain a colloid osmoticpressure that is sufficient enough to have a sufficient amount of bloodcirculating and establish an efficient enough blood pressure, the sizeof the gelatin molecules would be such that gelling becomes a problem.

To render gelatin suitable as a plasma expander, it has been chemicallymodified in such a way that gelability is drastically reduced. For thispurpose it is known that gelatin can be simultaneously degraded andcrosslinked, branched or inter-molecular bridges can be formed from thegelatin molecules. Probably the most successful modification is thepreparation of succinylated gelatin as described in U.S. Pat. No.2,827,419. A commercial preparation based on succinylated gelatin iscurrently available, known as Gelufusine®. The gelatin that is used isisolated from bovine origin and has an average molecular weight of30,000. Other commercially available modified gelatines are Geloplasma®(‘poligelatin’) and Gelifundol® (‘oxipoligelatin’).

A disadvantage of the presently used gelatin derivatives as colloidaladditives in plasma substitution compositions is the occurrence ofhypersensitivity reactions in subjects. In particular subjects having anallergy or an auto-immune disease, or for some other reason having anincreased level of antibodies, in particular IgE antibodies, are atrisk. A case of acute emergency in which the administration of plasmaexpanders is required is in subjects suffering from shock, more specifichypovolemic shock due to severe bleeding, excessive fluid loss orinadequate fluid uptake. In such a situation there is simply no time toassess possible risk factors, such as the presence of an allergy. If asubject is known to have an allergy, prophylactic administration of anantihistaminicum can be contemplated. However, in case of acuteemergency, any kind of prophylactic treatment is uncalled for. Thecondition of immediate hypersensitivity, which can occur uponapplication of the presently used gelatin derivatives, is known asanaphylactic shock. This is a life-threatening condition where bloodpressure is too low to sustain life, which in fact was the conditionthat should be counteracted by the plasma expander. Since a subjectreceiving the plasma expanders already suffers an acute trauma thecondition of anaphylactic shock is most likely to be fatal.

Another disadvantage of the commercially used gelatin derivatives is thefact that the gelatin used is isolated from animal sources such asanimal bone and hide, in particular it is derived from bovine sources.Disadvantages of this material are the presence of impurities and thefact that the nature of the composition is not clearly defined and thusnot reproducible. This may impose additional screening to ensure thatthe derivatisation process results in a product with the desiredproperties and may require careful purification steps. An additionalproblem nowadays, especially in relation to gelatin isolated from bovinesources, is the risk of contamination of the gelatin with factorsresposible for the occurence of Bovine Spongiform Encephalitis (BSE).For this reason the use of gelatin in blood substitution products may beprohibited. At present at least for one product, a modified gelatin ofbovine origin, it is known that as a precautionary measure the productis no longer commercially available.

Another disadvantage of the commercially used gelatin derivatives is thefa that the preparation of the gelatin figments with the intended sizedoes not result in fully homogeneous material but in a heterogeneousmixture of gelatin fragments around a targeted average molecular weight.The smaller fragments will leave the blood circulation system by anearly (unwanted) clearance by which their contribution to a stableclinical pattern is absent and the nephrotic system is negativelyimposed.

SUMMARY OF THE INVENTION

It is an object of the invention to provide alternative compositionssuitable as plasma substitution comprising a plasma expander, which willreduce the occurrence of immunological reactions, in particularanaphylactic shock.

Surprisingly it has been found that recombinant gelatin-like proteinswhich are in essence free of hydroxyproline do not give rise to animmunological reaction with blood samples containing IgE antibodies.

Thus, compositions as defined in the appending claims meet the objectiveof the invention, such a composition comprising a solution of saline ina physiologically acceptable concentration and a protein having acolloid osmotic function characterized in that the compound having aprotein colloid osmotic function is a recombinant gelatin-like proteinwhich is in essence free of hydroxyproline. Preferably the recombinantgelatin-like protein is also free of hydroxylysine; in additionpreferably it is also free of lysine.

The invention relates also to the use as a plasma expander of arecombinant gelatin-like protein which is in essence free ofhydroxyproline. Preferably for this use the recombinant gelatin-likeprotein is also free of hydroxylysine; in addition preferably it is alsofree of lysine.

DESCRIPTION OF THE INVENTION

According., to the invention a composition is provided comprising as acompound having a colloid osmotic function a recombinant gelatin-likeprotein which is in essence free of hydroxyproline.

Recombinant production of gelatin-like proteins in particular in amicro-organism allows reproducible production of proteins of constantcomposition without the risk of prion related health hazards.

In example 2 it is shown that in the case of two blood samples out of apanel of 60 samples obtained from subjects in which IgE antibodies inthe samples are present, two tested commercial preparations displayspecific binding of the IgE to the gelatin derivative, whereas in allthe samples the compositions according to the invention display no riskof a hypersensitivity reaction. If the subjects from which the twopositively tested samples originated, when in need, were to receive thecommercially available gelatin based plasma substitution compositions,said subjects would likely suffer anaphylactic shock.

A natural gelatin molecule in its primary amino acid sequence basicallyconsists of repeats of Gly-Xaa-Yaa triplets, thus approximately onethird of the total number of amino acids is a glycine. The molecularweight of gelatin is typically large, values of the molecular weightvary from 10,000 to 300,000 Daltons. The main faction of natural gelatinmolecules has a molecular weight around 90,000 Daltons. The averagemolecular weight is higher than 90,000 Daltons.

Furthermore, characteristic for gelatin is the unusual high content ofproline residues. Even more characteristic is that in natural gelatin anumber of the proline residues is hydroxylated. Most prominent site ofhydroxylation is the 4-position resulting in the presence in the gelatinmolecule of the unusual amino acid 4-hydroxyproline. In a triplet4-hydroxyproline is always found in the Yaa position. Very few prolineresidues are hydroxylated at the 3 position. In contrast with4-hydroxyproline, 3-hydroxyproline is always found at the carboxyl sideof a glycine residue, thus in the Xaa position in a triplet. Differentenzymes are responsible for the formation of 3- or 4-hydroxyproline.

Based on known amino acid compositions, it is estimated that in agelatin molecule derived from a mammal, approximately 22% of the aminoacids are a proline or a hydroxyproline residue. However lower contentsof proline and hydroxyproline are found in fish, in particular coldwater fish. A rough estimate is that proline and hydroxyproline residuesare present in approximately equal amounts, thus in a gelatin moleculederived from a mammal approximately 11% of the amino acids are prolinesand approximately 11% are hydroxyprolines. As substantially allhydroxyproline is found in the Yaa position, it is estimated thatapproximately one third of all triplets in a gelatin molecule comprise ahydroxyproline. The presence of the hydroxyproline residues isresponsible for the fact that a gelatin molecule in its secondarystructure can adopt a helical conformation.

Furthermore, another amino acid present in natural gelatin that is foundin very few other proteins is 5-hydroxylysine. Lysine residues modifiedin this way are always found in the Yaa position in a triplet.

Gelatin-like proteins for use according to the invention are understoodas proteins in which at least 5% of the total number of amino acids is aproline residue. By this percentage the gelatin-like characteristics,for the purpose of this invention not being defined as the gellingproperty but as the absence of unpreferred 3-dimensional globulardomains, is assured. Preferably in the gelatin-like protein at least10%, more preferably at least 15% of the total number of amino acids isa proline residue. The lower the proline content of a protein the morethe distribution of the proline residues in the protein becomesrelevant. Thus in a protein in which 5% of the total number of aminoacids is a proline residue, these residues are preferably evenlydistributed. In designing a suitable protein the skilled person, forinstance with the aid of computer modeling systems, will be able todesign sequences comprising proline residues which will not give rise toglobular domains. In order to prevent the formation of globular domainsas a guideline the gelatin-like protein for use in the inventionpreferably should not comprise stretches of more than 20 amino acidswithout a proline residue.

A predominant feature of gelatins is the presence of Gly-Xaa-Yaatriplets. Such triplets are preferably also present in the gelatin-likeproteins used in the invention. It is however possible to design aprotein in which Gly-Xaa-Yaa triplets or stretches of Gly-Xaa-Yaatriplets are separated by one or more amino acids. In such agelatin-like protein having ‘interrupted’ triplets or stretches oftriplets the definition of gelatin-like characteristics given above isuseful. In relation to a protein consisting completely of Gly-Xaa-Yaatriplets the definition given above of a gelatin-like protein for use inthe invention can be described as a protein in which at least 15% of thetriplets comprise a proline residue. Preferably such a gelatin-likeprotein does not comprise a stretch of more than 6 triplets without aproline residue. It is preferred a gelatin-like protein for use in theinvention comprises stretches of at least 10, preferably at least 20,more preferably more than 30 consecutive repeats of Gly-Xaa-Yaatriplets.

In order to maintain a suitable colloid osmotic pressure in combinationwith a targeted clearance rate from the blood circulation system whenadministered to a subject, the molecular weight of a gelatin-likemolecule for use according to the invention should be at least 10,000Daltons, preferably more than 15,000 Daltons, more preferably more than20,000 Daltons. Most preferably the molecular weight is between about30,000 Daltons and 80,000 Daltons. The gelatin-like molecule for useaccording to the invention is in essence free of hydroxyprolineresidues, meaning that less than 2% of the aminoacid residues in thegelatin-like protein are hydroxyproline residues, preferably less than1%. Preferably it is in essence free of hydroxylysine residues, meaningthat less than 0.2% of the aminoacid residues in the gelatin-likeprotein are hydroxylysine residues, preferably less than 0.1%.Advantageously it is in essence also free of lysine residues, meaningthat less than 2%, preferably less than 1% of the aminoacid residues inthe gelatin-like protein are lysine residues.

The amount of hydroxyprolines, hydroxylysines and lysines can bedetermined by any standard aminoacid analysis method like, for example,described in HP AminoQuant Series II, operators handbook, 1990,Hewlett-Packard GmbH, Federal Republic of Germany, Waldbronn AnalyticalDivision, HP Part No. 01090-90025.

For a considerable number of subjects gelatin and gelatin derivativesare not considered to be immunogenic compounds. For instance this isevidenced by the existence of commercial plasma substitution productsbased on gelatin. The fact that the gelatin that is used is not human inorigin appears to be no problem. Also the fact that such gelatins arechemically modified appears not to be a problem. A sufficient amount ofsubjects are potentially able to benefit from the presently commonpreparations, giving such preparations reason to exist.

There are however subjects, like those having allergies or auto-immunediseases, that cannot tolerate the commercial preparations for plasmasubstitution based on gelatin derivatives. Faced with this problem afirst improvement could be to try to improve on the purification of thegelatin proteins. One approach is optimizing even further the isolationprocedure of natural gelatin or optimizing the derivatization andsubsequent purification procedure. Another possibility could lie inalternative sources or alternative production methods for gelatin.Having knowledge of the current biotechnological developments and theadvance that is made with respect to recombinant production of gelatinsand collagens it could be contemplated to follow such a route forreproducible production of proteins of constant composition.

As mentioned earlier, although gelatins appear not to be veryimmunogenic they can be lethal to subjects having an allergy or anauto-immune disease. When pursuing the approach of recombinantproduction of gelatins and bearing in mind that such gelatins should beeven less immunogenic in human subjects than the presently used bovinederived gelatins, it is obvious to take up production of recombinanthuman gelatin. In addition it is obvious not to induce marked changes inthe basic gelatin structure.

In contrast to these obvious possible solutions, suitable gelatin-likeproteins for use according to the invention can be non-natural, or canbe equivalent to natural occurring gelatins. Non-natural in this contextmeans that the gelatin is derived from a synthetic gene. The mostprominent difference of the gelatin-like proteins for use according tothe invention is the absence of hydroxyproline residues compared tonatural gelatin molecules. The presence of hydroxyproline residues innatural gelatin allows the molecule to adopt a helical conformation. Theabsence of hydroxyproline residues prevents the gelatin-like proteinsfrom adopting such a conformation and prevents the gelatin-like moleculefrom gelling, even at low temperatures.

There is no prior art information on immunological or antigenicproperties of gelatin-like proteins useful in the invention. Thedistinctiveness of the gelatin-like proteins for use according to theinvention from natural gelatin, both chemically and conformationally,would dissuade the use of such a protein in pleura substitutioncompositions. Surprisingly however, gelatin-like proteins for useaccording to the invention show no immunogenic interaction with bloodhaving increased amounts of IgE antibodies.

Gelatin-like proteins for use according to the invention are in essencefree of hydroxyproline. This means that to a certain level ofhydroxyproline residues is allowed. The level of hydroxyproline residuesshould be lower than the minimum level that is required to let a 5weighty solution of the protein in isotonic saline at neutral pH gel at5° C. This condition is met by for instance gelatin-like proteins inwhich less than 2% of the aminoacid residues are hydroxyprolineresidues.

In a further embodiment, the gelatin-like protein for use in theinvention is in essence free of hydroxylysine. This is most efficientlyachieved by avoiding the formation of hydroxylysine in yet a furtherembodiment in which the gelatin-like protein for use in the invention isin essence free of lysine. As mentioned above, hydroxylysine is rarelyfound in proteins. It could be possible that hydroxylysine or aparticular sequence in which hydroxylysine is present, is involved inantigenic interactions. Antigenic interactions could be the result ofthe presence of the amino acids itself or could be the result ofparticular conformations the gelatin or gelatin derivative might adoptdue to the presence of hydroxylysine.

The gelatin-like protein can be made de novo from a synthetic nucleicacid sequence. This allows tailor-made design of the protein. Thedesigned synthetic nucleic acid sequence can be expressed in suitablemicro-organisms using known recombinant techniques.

With respect to the design of gelatin-like proteins for use in theinvention, several properties of the proteins are addressed. Forinstance it can be made sure specific amino acids, such as lysine, willnot occur in the protein. Otherwise, as discussed below in particularwith respect to lysine as well, it can be advantageous to introduce adefinite number of a specific amino acid in the gelatin-like protein.Also the clearance speed of the gelatin-like proteins can be“designed-in” by the choice for a or size or a specific range of sizesof the gelatin-like proteins. In particular this could be advantageousin combination with known nephrotic system characteristics (measured byfor instance the creatinine clearance pattern) of subjects to whom thegelatin-like proteins are administered. The size of the gelatin-likeproteins is further of importance for the colloid osmotic pressure, asdiscussed below, it exercises. Yet further the iso-electric point (IEP)can be tuned by the composition of acidic and basic amino acid residuesin the gelatin-like proteins.

In one embodiment the composition according to the invention comprises agelatin-like protein which is homodiperse in nature. Homodisperse meansof constant composition and molecular weight. Variations in compositionthat can occur due to the recombinant production process are allowed. Interms of molecular weight a useful definition of homodispersity would bethat at least 90% of the total amount of gelatin-like protein in thecomposition has a molecular weight that lies within a rage of plus orminus 10% around a selected molecular weight. The selected molecularweight depends on the desired colloid osmotic pressure and on thedesired clearance rate from the blood circulation system. In anotherembodiment the composition according to the invention comprises two ormore gelatin-like proteins each being homodiperse in nature but withdifferent molecular weights. The difference in molecular weight resultsin a different clearance pattern from the circulating blood. Such acomposition allows tuning of the plasma expanding activity of thecomposition over prolonged periods of time.

The starting point for the gelatin-like protein for use in the inventioncan also be an isolated gene encoding a naturally occurring gelatinmolecule, which is processed further by recombinant means. Preferablythe gelatin-like protein used according to the invention resembles ahuman native amino acid sequence with this difference that in essencehydroxyproline residues are absent.

The gelatin-like proteins for use according to the invention can beproduced by recombinant methods as disclosed in EP-A-0926543 andEP-A-1014176. For enablement of the production and purification ofgelatin-like proteins that can be suitably used in composition accordingto the invention specific reference is made to the examples inEP-A0926543 and EP-A-1014176. Thus the gelatin-like proteins can beproduced by expression of nucleic acid sequence encoding suchpolypeptide by a suitable micro-organism. The process can suitably becarried out with a fungal cell or a yeast cell. Suitably the host cellis a high expression host cells like Hansenula, Trichoderma,Aspergillus, Penicillium, Neurospora or Pichia. Fungal and yeast cellsare preferred to bacteria as they are less susceptible to improperexpression of repetitive sequences. Most preferably the host will nothave a high level of proteases that attack the collagen structureexpressed. In this respect Pichia offers an example of a very suitableexpression system. As disclosed in EP-A0926543 and EP-A-1014176specifically Pichia pastoris is used as expression system. Preferablythe micro-organism is fee of active post-translational processingmechanism such as in particular hydroxylation of proline and alsohydroxylation of lysine. The host to be used does not require thepresence of a gene for epression of prolyl-4-hydroxylase. Preferably thehost also does not require the presence of lysyl-hydroxylase. Generallythe recombinant production method will result in proteins comprisingnatural amino acids, i.e. L-amino acids. The presence of D-amino acidsas a result of isomerisation processes that can occur naturally isallowed. Less than 1% of the amino acids is in the D-form. The selectionof a suitable host cell from known industrial enzyme producing fungalhost cells specifically yeast cells on the basis of the requiredparameters described herein rendering the host cell suitable forexpression of recombinant gelatin-like proteins suitable in compositionsaccording to the invention in combination with knowledge regarding thehost cells and the sequence to be expressed will be possible by a personskilled in the art.

When produced by recombinant means, especially by expression ofrecombinant genes in yeasts, the proteins for use according to theinvention preferably do not contain cysteine or another mercapto aminoacid, nor do they contain a combination of methionine and arginine in 14position (Met-Xay-Xaz-Arg), as such a sequence is sensitive to enzymaticproteolysis.

It may be noted that the proteins for use according to the invention canalso be partly or wholly produced by methods other than DNA expression,e.g. by chemical protein synthesis; in that case, they may also containnon-natural amino acids.

In order to obtain the composition of the invention the gelatin-likeprotein is dissolved in saline in a physiologically acceptableconcentration at physiological pH. Saline is a solution of Na⁺ and Cl⁻ions in water. Since it is highly likely that plasma substitutioncompositions are administered in great volumina, care should be takenthat dilution effects do not disturb electrolyte balances. Whenpreparing compositions according to the invention the skilled personwill be able to apply appropriate concentrations of Na⁺ and Cl⁻ ions.Workable margins would be 120-170 mmol/l for Na⁺ and 90-140 mmol/I forCl⁻. If so desired the composition according to the invention couldcomprise one or more additional components normally found in blood. Forinstance a composition according the invention comprises one or morecomponents in a physiologically acceptable concentration selected fromMg²⁺, K⁺, Ca²⁺, HPO₄ ²⁻, H₂PO₄ ⁻ and glucose. The skilled person will beable to determine what is a physiologically acceptable concentration foreach component. Suitably, a composition according to the invention alsocomprises a buffering compound, preferably selected from the groupconsisting of HCO₃ ⁻ and lactate. The skilled person will be able todetermine the appropriate amount of buffer in order to maintain thecomposition at a physiologically acceptable pH.

It is preferred the composition according to the invention isapproximately isotonic or iso-osmotic with blood of human subjects,therefore the composition has an osmolarity preferably in the range from270-300 mOsm.

The purpose of the gelatin-like proteins is to maintain an appropriatecolloid osmotic pressure in order to keep a sufficient amount of bloodvolume circulating. The non-gelling property of the proteins for useaccording to the invention has the advantage that macromolecules ofconsiderable size can be used which will not be rapidly cleared from thesystem. In order to be effective as plasma expander the gelatin-likecompounds should have a molecular weight of at least 10,000 Daltons,preferably at least 15,000 Daltons, even more preferable at least 20,000Daltons. Most preferably the molecular weight is between about 30,000Daltons and 90,000 Darns. According to the invention it is possible toapply even much larger gelatin-like proteins in case this is preferreddepending on the desired colloid osmotic pressure and/or the desiredclearance rate from the blood circulation system. Compositionscomprising gelatin-like proteins of high molecular weight can be appliedwithout the risk of gelling or of a too high viscosity. It does not seemlikely however, that gelatin-like proteins having a molecular weight ofhigher than 100,000 Daltons can be suitably applied in compositionsaccording to the invention.

The composition of the invention comprises an amount of gelatin-likeproteins which exerts an osmotic pressure comparable to or slightlyexceeding the osmotic pressure exerted by human serum albumin in blood.Determining the colloid osmotic pressure of a composition is a routinemeasurement for the skilled person, for instance by using a commerciallyavailable membrane osmometer equipped with a suitable semi-permeablemembrane, for instance with a cut-off of 20,000 Daltons. The skilledperson will be able to determine the correct amount of gelatin-likeprotein suited for the desired osmotic pressure. Usually the amount ofgelatin-like protein that can be applied lies in the range from 2-8weight %.

If so desired, it is possible to introduce simultaneously with theplasma substitution composition of the invention a pharmacologicallyactive compound. For instance it may be advantageous to simultaneouslyintroduce medicaments involved in the blood clotting process. Inparticular such a composition could be of use in the application of aplasma expander during surgery or preoperative dilution of blood. Thusin another embodiment the composition according to the inventioncomprises a pharmacologically active compound.

Making use of the advantageous property of the gelatin-like protein thatit has a sustained circulation time in plasma it is particularlyenvisaged to covalently attach pharmaceutically active compounds to thegelatin-like protein. In a further embodiment the composition accordingto the invention comprises a pharmaceutically active compound which iscovalently attached to the gelatin-like protein.

Covalent attachment of a pharmaceutically active compound to a proteinis routine practice for an ordinary skilled organic chemist. Forinstance coupling of a carboxyl function in a drug to an amino group ofa lysine in a protein can be achieved by converting the carboxyl groupin its activated ester using DCC, or EDC, and NHS, which reacts with thefree amine.

As in a protein lysine residues are the residues of choice for thecovalent attachment of other molecules, it is for this purpose notdesired to have a protein that is in essence free of lysine residues. Incontrast, lysine residues should be present and preferably the number oflysine residues present is known, for this allows an estimation of howmany pharmacologically active compounds are coupled to a protein andthus allows appropriate dosage of the medicament. The design ofsynthetic nucleic acid sequences de novo now offers the advantageouspossibility to introduce a specific amount of lysine residues and thusthe production of well defined gelatin-like proteins bearingpharmaceutically active compounds. A distinct correlation betweenclearance time of the protein and dosage of the medicament can be made.

After administration the coupled medicament will not diffuse from thecirculating blood into the interstitium. This is a specific advantagefor medicaments which should function intravascularly. Unwanted sideeffects by diffusion of the medicament into the interstitial fluidthroughout a subject are avoided. Also medicaments having anintravascular as well as an extravascular activity profile could benefitfrom the focus on the intravascular mode of action.

Clearance by liver and kidney will be kept to a minimum ensuring a moreconstant plasma level of the medicament. Half-lives of medicamentscoupled to gelatin-like proteins will be increased.

Examples of medicaments which are administered intravascularly and whichare suitable for coupling to the protein used in the invention aremedicaments involved in intervening blood clotting, vasodilatation,function of erythrocytes, thrombocytes and leukocytes, thrombosis,immuneresponses, blood levels of messenger molecules such as hormonesSpecific examples are heparin, beta-blockers, blood pressure regulatorssuch as angiotensin antagonists and antibiotics.

It should be understood that modification of the gelatin-like proteinsfor use in compositions according to the invention is not restricted tothe coupling of pharmacologically active compounds. To improve theproperties other modifications after the gelatin-like protein has beenrecombinantly produced and isolated are possible. For instancemodifications to influence the iso-electric point or the solubility oranother relevant property can be advantageous. Care should be taken thatsuch a modification does not introduce elements that are likely toinduce an immunogenic or antigenic reaction.

DESCRIPTION OF THE FIGURES

FIG. 1.: Plasma volume expansion as a function of time after infusion ofbiogel-I.

FIG. 2.: Plasma volume expansion as a function of time after infusion ofGelifundol.

FIG. 3.: Volume expansion by infused colloid at t=60 minutes.

FIG. 4.: Gelatin plasma concentration as a function of time afterinfusion of Biogel-I or Gelifundol.

EXAMPLES Example 1 Recombinant Collagen-Like Peptide

General Molecular-Biological Techniques

Cloning procedures were performed essentially according to Maniatis etal. [Maniatis T., Fritsch, E. F. & Sambrook, J. (1982) Molecularcloning: A laboratory manual. Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.]. Plasmid DNA was isolated using Wizard Plus SV miniprep,or Qiagen miniprep systems. DNA was isolated from agarose gels using theQIAquick Gel Exaction Kit (Qiagen). All enzymes used were from AmershamPharmacia Biotech unless otherwise stated and were used according to therecommendations of the manufacturer. AU procedures involving thehandling and transformation of Pichia pastoris were essentiallyperformed according to the manual of the Pichia Expression Kit(Invitrogen) [Manual of the Pichia Expression Kit Version E (Invitrogen,San Diego, Calif., USA)].

Construction of pPIC9-H1

A synthetic gene encoding a hydrophilic gelatin with six histidineresidues (referred to hereafter as “Biogel-II”) was designed to have thecodon usage of Pichia pastoris highly expressed genes (Sreekrishna, K.and Kropp, K. E. (1996) Pichia pastoris, Wolf, K. (Ed), Non conventionalyeasts in biotechnology. A handbook, Springer-Verlag, pp. 6/203-6/253).

Two separate PCR reactions were performed, using the followingoligonucleotides:

-   1. 1 pmol OVL-PA-FW, 1 pmol OVL-PA-RV, 50 pmols HLP-PA-FW and 50    pmols HLP-PA-RV.-   2. 1 pmol OVL-PB-FW, 1 pmol OVL-PB-RV, 50 pmols HLP-PB-FW and 50    pmols HLP-PB-RV.

Oligonucleotide sequences were as follows: HLP-PA-FW: 5′-GCGCTCGAGAAAAGAGAGGC TGAAGC-3′ OVL-PA-FW: 5′-GCGCTCGAGA AAAGAGAGGC TGAAGCTGGTCCACCCGGTG AGCCAGGTAA CCCAGGATCT CCTGGTAACC AAGGACAGCC CGGTAACAAGGGTTCTCCAG GTAATCCCA-3′ OVL-PA-RV: 5′-TGAGAACCTT GTGGACCGTT GGAACCTGGCTCACCAGGTT GTCCGTTCTG ACCAGGTTGA CCAGGTTGAC CTTCGTTTCC TGGTTGACCTGGATTACCTG GAGAACCCTT-3′ HLP-PA-RV: 5′-TGAGAACCTT GTGGACCGTT GGAA-3′HLP-PB-FW: 5′-TTCCAACGGT CCACAAGGTT CTCA-3′ OVL-PB-FW: 5′-TTCCAACGGTCCACAAGGTT CTCAGGGTAA CCCTGGAAAG AATGGTCAAC CTGGATCCCC AGGTTCACAAGGCTCTCCAG GTAACCAAGG TTCCCCTGGT CAGCCAGGTA ACCCT-3′ OVL-PB-RV:5′-GCGTCTGCAG TACGAATTCT ATTAGCCACC GGCTGGACCC TGGTTTCCTG GTTTACCTTGTTCACCTGGT TGACCAGGGT TACCTGGCTG ACCAGGGGAA CCTTGGTT-3′ HLP-PB-RV:5′-GCGTCTGCAG TACGAATTCT ATTAGC-3′

The 50 μl PCR reactions were performed in a GeneAmp 9700 (Perkin-Elmer)and contained 0.2 mM dNTP's Pharmacia), 1× Pwo buffer (Eurogentec) and1.25 U Pwo polymerase (Eurogentec). Reaction 1 involved 18 cyclesconsisting of 15 seconds at 94° C. and 15 seconds at 72° C. Reaction 2involved a touchdown PCR, whereby each cycle consisted of 15 seconds at94° C., 15 seconds at the annealing temperature and 15 seconds at 72° C.The annealing temperature was lowered from 72° C. to 68° C. in the first5 cycles, after which 20 additional cycles at an annealing temperatureof 67° C. were performed.

The PCR products were isolated from agarose gel. 0.3 pmols of eachfragment and 50 pmols of the outer primers HLP-PA-FW and HLP-PB-RV weresubjected to overlap extension PCR. 25 cycles consisting of 15 secondsat 94° C., 15 seconds at 67° C. and 15 seconds at 72° C. were performed.The resulting 0.3 kb PCR fragment was digested with XhoI/EcoRI andinserted in cloning vector pMTL23. An errorless clone (referred tohereafter as “pMTL23-P”) was selected by verification of the sequence byautomated DNA sequencing.

An additional PCR reaction was performed using the followingoligonucleotides: 1 pmol OVL-H-FW, 1 pmol OVL-H-RV, 50 pmols HLP-H-FWand 50 pmols HLP-H-RV.

Oligonucleotide sequences were as follows: HLP-H-FW: 5′-CCACCCGGTGAGCCAGGA-3′ OVL-H-FW: 5′-CCACCCGGTG AGCCAGGAAA CCCTGGTCAC CACGGTAACCAAGGACAGCC AGGTAACGAA GGTCAACCAG GTCAGGAAGG TAATCCTGGA AACGAGGGTC AT-3′OVL-H-RV: 5′-GCCACCGQCT GGACCTTGGT TACCGTGGTG TCCCTGCTCA CCAGGTTGACCTGGTTGACC CTCGTTTCCA GGTTGACCGT GATGACCCTC GTTTCCAGGA TT-3′ HLP-H-RV:5′-GCCACCGGCT GGACCTTG-3′

The 50 μl PCR reactions were performed in a GeneAmp 9700 (Perkin-Elmer)and contained the oligos indicated above and 25 μl of High Fidelity PCRMaster (Roche).

The reaction involved 18 cycles consisting of 15 seconds at 94° C., 15seconds at 60° C. and 15 seconds at 72° C. The 0.18 kb PCR product wasisolated from agarose gel and T/A cloned into vector pGEM-T Easy(Promega). An errorless clone was selected by verification of thesequence by automated DNA sequencing. The vector was then digested withDraIII/Van91I. The resulting 0.18 kb fragment was isolated from agarosegel and cloned into Van91I digested, dephosporylated pMTL23-P. Theresulting vector was cut with EcoRI/hol, after which the insert wascloned into EcoRI/XhoI digested P. pastoris expression vector pPIC9, toyield vector pPIC9-H1.

The encoded amino acid sequence of the mature (processed) Biogel II isas follows: 1 G P P G E P G N P G S P G N Q G Q P G N K G S P G N P G QP 31 G N E G Q P G Q P G Q N G Q P G E P G S N Q P Q G S Q G N P 61 G KN G Q P G S P G S Q G S P G N Q G S P G Q P G N P G Q P 91 G E Q G K P GN Q G P A G E P G N P G H H G N Q G Q P G N E 121 G Q P G Q E G N P G NE G H H G Q P G N E G Q P G Q P G E Q 151 G H H G N Q G P A G G

Molecular weight: 15.1 kDa, isoelectric point: 5.1.

Example 2 Radio Allergen Sorbent Test (RAS Test or RAST)

In order to demonstrate the presence of IgE antibodies against certainallergens or proteins the RAS test is used. For a detailed descriptionof the RAS test reference is made to Aalberse et al. J. Allergy Clin.Immunol., 1981, vol. 68: 356-364.

The compositions which contain gelatines that are tested are:

-   Gelofusine®, Gelifundol®, Composition containing Biogel-I,    Composition containing Biogel-II and Composition containing    Biogel-III.

Gelofusine® (modified gelatin 40 g/l Na⁺ 154 mmol/l, Cl⁻ 125 mmol/l) andGelifundol® (modified gelatin 55 g/l, Na⁺ 145 mmol/l, Cl⁻ 100 mmol/l,NaEDTA 0.19 g/l, Ca²⁺ 0.5 mmol/l, HCO₃ ⁻ 30 mmol/l) were used ascommercially obtained.

Biogel-I and Biogel-III are described in EP-A-0926543 FIG. 3 andEP-A-1014176 page 7, respectively. Biogel-II is decribed in example 1.Compositions comprising 55 g/l gelatin-like proteins Biogel-I, Biogel-Iland Biogel-III in PBS Na⁺ 164 mmol/l. Cl⁻ 140 mmol/l, HPO₄ ⁻ 10.9mmol/l, H₂PO₄ ²⁻ 1.8 mmol/l were prepared.

Sera of subjects which are known to have an allergy against specificfoodstuffs were tested. The sera were selected on the known presence ofIgE antibodies against foodstuff, in particular against beef, pork andegg. Subjects having IgE antibodies against these foodstuffs possiblyalso have IgE antibodies against gelatin.

In addition 49 plasma samples obtained from plasmafereses, selected onthe presence of IgE antibodies against known allergies, were tested.

The gelatin derivative or gelatin-like protein is conjugated toCNBr-activated Sepharose beads (Amersham Pharmacia Biotech, Uppsala,Sweden) (approximately 1 μg protein per mg beads) following a standardconjugation protocol according to the manufacturer's instructions.

Using a buffer containing Human Serum Albumin the concentration isadjusted to 2 mg beads per ml.

250 μl Sepharose beads conjugated to a gelatin derivative or agelatin-like protein are incubated overnight at room temperature with 50W serum or plasma sample.

The beads are washed 4 times to remove excess serum or plasma andresuspended in 250 μl medium.

The beads are incubated overnight at room temperature with 50 μlanti-human IgE antibody labeled with ¹²⁵I. The labeled IgE antibody isprepared following a standard procedure using chloramine T.

The beads are washed 4 times to remove excess ¹²⁵I labeled anti-humanIgE antibody. The reactivity in the samples is counted (with positiveand negative controls). The presence of reactivity in a sampledemonstrates binding of IgE in a serum or plasma to the gelatinderivative or gelatin-like protein and thus the risk of the occurrenceof a hypersensitivity reaction.

Results Biogel- Biogel- Gelofusine ® Gelifundol ® Biogel-I II III Serum++ ++ −− −− −− 3093 Serum PF ++ ++ −− −− −− 175 Other sera −− −− −− −−−−++ = specific immune reaction−− = no immune reaction

In a control experiment the samples tested positive are pre-incubatedwith Gelofusine® or Gelifundol®. After pre-incubation, in the RAS testno radioactivity is found. The immunological reaction is specific forthe gelatin derivatives used.

Preclinical Evaluation of Gelatin Solutions in Rats

In a preclinical evaluation of recombinant gelatin solutions in rats thefollowing is addressed:

-   -   the capacity to expand the vascular volume (primary        pharmacologic property)    -   the distribution, plasma half-life and excretion through the        kidneys (pharmacokinetics)

Filling of the vascular system is related to the oncotic activity invivo. Within limits, the amount of macromolecules infused determines themagnitude of the effect and not the concentration in the infusedsolution. The oncotic activity can therefore be determined by studyingthe hemodilution by a certain dose of gelatin. The accuracy will bebetter when different doses are applied. In practice, to obtain goodmeasurable effects, for example, 20 ml/kg bodyweight (about 30% of theblood volume) can be withdrawn and replaced by the same volume ofsolutions with different concentrations of gelatin (around the estimatediso-oncotic concentration). The in vivo iso-oncotic concentration can bedetermined by comparing the actual effects on the red blood cell countwith the expected effects.

When the macromolecules are cleared from the circulation the plasmavolume will decrease, leading to an increase in red blood cell count orhematocryt. Therefore, measuring the changes in the red blood cell countand the gelatin plasma concentration in time will reveal the duration ofthe effect and the half-life of gelatin in the circulation. Whendifferent doses are applied it will become clear whether or not thehalf-life is dose dependent within the range of clinically relevantdoses. Relatively small macromolucules (<30 kD, depending on charge andshape) may be cleared by the kidneys. Kidney excretion can be determinedby collecting urine and measuring the gelatin concentration. When largeamounts of gelatin are excreted, kidney tubuli may become blocked byprecipitation of gelatin in kidney tubuli. This can be studied by lightmicroscopy.

Constituents of the gelatin solutions, especially impurities from yeast,may induce inflammatory responses. This may, amongst others, lead tovasoactivity and/or activation of neutrophils.

Because the half-life is probably in the order of hours, a 4 hourduration seems to be sufficient for initial experiments. This means thatthe whole experiment can be done under anesthesia, which facilitatesblood pressure measurement and blood sampling and minimizes discomfortfor the rats.

The iso-oncotic activity can be determined without measuring plasmaconcentrations, but for determination of the clearance an assay formeasuring gelatin in plasma and urine should be available.Alternatively, labeled gelatin could be used, with the drawback thatlabeling may change the properties.

Protocol:

Animal Data

-   Species: Rat-   Strain/Sex: Wistar HsdCpb:WU, female    Procedures-   1. Administration of Testsolutions-   Withdrawal of blood: 20 ml/kg in 10 minutes-   Infusion of gelatin solution: 20 ml/kg (4-6 ml) in 10 minutes-   2. Blood and Urine Samples-   Blood: 0.2 to 1.5 ml blood samples were collected from the venous    cannula into syringe and rapidly transferred into EDTA-containing    polypropylene vials at t=0, 60, 120 and 240 min.-   Urine was collected into a preweighted vial. The volume was    determined by weighing.-   3. Duration of the Experiment

The experiments were terminated 240 minutes after administration of thetest solution by giving a lethal dose of pentobarbital.TEST/CONTROL/COMPARISON SOULTION Test solution 1 recombinant ratgelatin, 36 kD Biogel-I identity Biogel-I 36 kD supplier Fujiformulation freeze dried remarks reconstituted with 0.9% NaCl at either3 or 4 g/ 100 ml and stored at 4° C. until adminstration (for less than1 week) Control solution saline identity 0.9% (w/v) NaCl supplier NPBI,Emmer-Compascuum, The Netherlands formulation sterile fuid for ivadministration remarks Comparison solution 1 human albumin identityCealb supplier CLB formulation solution for iv infusion, 20 g/100 mlremarks stored at 4° C., diluted with saline to 5 g/100 mL Comparisonsolution 2 modified bovine gelatin identity Gelifundol supplier BiotestPharma GmbH formulation solution, 5.5 g/100 ml remarks stored at 4° C.,diluted with saline to 4 g/100 mLLaboratory Investigation

-   a) Hematocrit was measured by centrifugation of blood in glass    capillaries at 10.000 g for 5 min.-   b) Red Blood cell count was done with an electronic cell counter    (model ZF; Coulter Electronics)-   c) Measurement of gelatin concentrations-   Gelatin conc were determined using reversed phase chromatography and    detection at 220 nm    Calculations

The hematocrit at each time point is calculated from the rbc count atthat time point, the rbc count at t=0 and the hematocrit at t−0.

The expected (hypothetical) volumina are calculated as follows:

-   i) expected blood volume (BV) is calculated assuming that no fluid    shifts occur:-   at t=0 in ml: 65 (ml/kg)*body weight (kg)-   at t≧20: BV t=0−withdrawn volume+infused volume-   ii) expected plasma volume (PV) is calculated in ml assuming that no    fluid shifts occur and that the body hematocrit is equal to that in    the peripheral blood:-   at t=0 in ml: BV t=0*(1-hct t=0)-   at t≧20: PV t=0−withdrawn plasma+infused volume-   iii) expected hematocrit is calculated as (BV-PV)/BV

The real volumes are estimated as follows,

-   i) estimated real BV at t-0 as expected thereafter estimated from    the ratio between the expected and the observed hematocrit:-   at t=0 in ml: 65 (ml/kg)*body weight (kg)-   at t≧20: BV expect*exp hct/obs hct-   ii) estimated real PV at t=0 as expected thereafter estimated from    claculated real BV and the observed hematocrit:-   at t=0 in ml: BVt=0*(1-hct t=0)-   at t≧20: estimated real BV*(1-hct)

The volume expansion by the infused test solution at t-60 was estimatedfrom the infused volume and the difference between the estimated realplasma volume and the expected plasma volume:

-   i) volume expansion at t=60: infused volume−expected    PVt=60+estimated real PVt=60-   ii) volume expansion per g colloid (ml/g)    Results and Discussion    Expansion of Plasma Volume

FIG. 1 shows that withdrawal of blood and subsequent saline infusionresulted in an initial decrease of the hematocrit to about the expectedvalue. However, thereafter hematocrite rose to substantially highervalues. After 5% Albumin infusion the hematocrit values remained belowthat expected throughout the observation period. Infusion of Saline,without oncotic activity, resulted in hypovolemia in this model, whereas5% Albumin induced sustained hypervolemia, which means that it ishyperoncotic for the rats. The volume expansion induced by the infusionof 30 ml/kg of infusate was calculated from the hematocrit changes. Forsaline this was 17-18 ml/kg, for 5% Albumin 40-44 ml/kg, both 1 hourafter infusion. For albumin, this means an expansion of 27-30 ml pergram albumin in this model.

Biogel-I was hyperoncotic at a concentration of 4 g/10 ml (FIG. 1),there was clear hypervolemia during 2 to 3 hours The plasma expansionwas 44-59/g after infusion of 30 ml/kg. 3 g/100 ml appeared to be closeto the isoncotic concentration, the plasma expansion 35-44 ml/kg.Overall, the volume expansion after 1 hour was 43 +/−6 (mean & SD) mlper gram of Biogel-I.

Gelifundol 4 g/100 ml had a very short volume effect (FIG. 2). Thevolume expansion after 1 hour was 17 and 25 ml/kg for an infused volumeof 30 ml/kg, which is hardly more than saline. The volume expansionafter 1 hour was 14-21 ml per gram colloid.

FIG. 3 compares the onctoic effect as volume expansion of Biogel-1 toGelifundol and HSA, showing the improved short tern volume expansion ofbiogel-I.

These results show that Biogel-I is a very effective plasma expanderboth in short term- and long term volume expansion. The in vivo oncoticeffect was, on a weight base, about 50% higher than that of humanalbumin. In comparison, Gelifundol had a minimal and very short-lastingoncotic effect in the rat model.

B. Plasma Clearance

FIG. 4 shows the gelatin plasma concentrations after infusion of 3 g/100ml and 4 g/100 ml Biogel-1 and 4 g/100 ml Gelifundol.

For Biogel-I, the initial distribution volume at t-20 min was 49-54ml/kg independent of the infused concentration. This corresponds more orless to the expected plasma volume, indicating intravasculardistribution without binding. The concentration then slowly decreased to20-30% of the initial value after 4 hours. From these curves an apparentplasma half-life was calculated for Biogel-I of 87 +/−13 min (mean &SD). It should be noted that in the 3 hour observation period alsodistribution occurs into the extravascular space. There was no excretiondetected into the urine.

Gelifundol had an initial distribution volume of 75-82 ml, suggestingthat part of the gelatin had already disappeared from the circulation inthe first 10 minutes after infusion. There was a relatively rapiddecrease of the plasma concentration, which was accompanied by asubstantial decrease in plasma volume in the period from t=20 to t=60min (see FIG. 2). The results indicate a very rapid dissappearance fromthe circulation, more than 50% disappeared within one hour and theplasma volume expanding effect lasts less than 20 minutes (FIG. 2). Highgelatin concentrations were present in the urine, suggesting that thekidney excretion is a major clearance mechanism.

These results show that immediately after intravenous administration,Biogel-I distributed into the plasma compartment. The benefit of theinvention plasma expander is shown since, In contrast to Gelifundol, itwas not excreted into the urine. The plasma half-life is 1 tot 2 hoursin the rat model. The effective period of volume expansion is therebyshorter than that of albumin, but much longer than that of Gelifundol.

1. Composition suitable as a substance for plasma comprising a solutionof saline in a physiologically acceptable concentration and a proteinhaving a colloid osmotic function wherein the protein having a colloidosmotic function is a recombinant gelatin-like protein comprisingGly-Xaa-Yaa triplets in which less than 2% of the amino acid residues inthe gelatin-like protein are hydroxyproline residues, preferably lessthan 1%.
 2. Composition according to claim 1 in which the gelatin-likeprotein is in essence free of hydroxyproline residues.
 3. Compositionaccording to claim 1 in which less than 0.2% of the amino acid residuesin the gelatin-like protein are hydroxylysine residues, preferably lessthan 0.1%.
 4. Composition according to claim 1 in which the recombinantgelatin-like protein is in essence free of hydroxylysine.
 5. Compositionaccording to claim 1 in which less than 2%, preferably less than 1% ofthe amino acid residues in the gelatin-like protein are lysine residues.6. Composition according to claim 1 in which the recombinantgelatin-like protein is in essence free of lysine.
 7. Compositionaccording to claim 1 in which the recombinant gelatin-like protein has amolecular weight of at least 10,000 Daltons, preferably at least 15,000Daltons, more preferably at least 20,000 Daltons.
 8. Compositionaccording to claim 7 in which the recombinant gelatin-like protein has amolecular weight between about 30,000 Daltons and 80,000 Daltons. 9.Composition according to claim 1 in which the recombinant gelatin-likeprotein is homodisperse.
 10. Composition according to claim 1 whichcomprises two or more recombinant gelatin-like proteins each beinghomodisperse but with a different molecular weight.
 11. Compositionaccording to claim 1 which comprises one or more components in aphysiologically acceptable concentration selected from Mg²⁺, K⁺, Ca²⁺,HPO₄ ²⁻, H₂PO₄ ⁻ and glucose.
 12. Composition according to claim 1 whichcomprises a buffering compound, preferably selected from HCO₃ andlactate.
 13. Composition according to claim 1 which comprises apharmacologically active compound.
 14. Composition according to claim 13in which the pharmacologically active compound is covalently attached tothe gelatin-like protein.
 15. Use as a plasma expander of a recombinantgelatin-like protein which is in essence free of hydroxyproline.
 16. Useaccording to claim 15 in which the gelatin-like protein is in essencefree of hydroxylysine.
 17. Use according to claim 15 in which thegelatin-like protein is in essence free of lysine.
 18. Compositionaccording to claim 1 in which the gelatin-like protein is in essencefree of hydroxyproline residues, in which less than 0.1% of the aminoacid residues in the gelatin-like protein are hydroxylysine residues orlysine residues and in which the recombinant gelatin-like protein is inessence free of hydroxy lysine.
 19. Composition according to claim 18 inwhich the recombinant gelatin-like protein has a molecular weight offrom about 30,000 Daltons to about 80,000 Dalton and is homodisperse andwhich comprises one or more components in a physiologically acceptableconcentration selected from Mg²⁺, K⁺, Ca²⁺, HPO₄ ²⁻, H₂PO₄ ⁻ andglucose.
 20. Composition according to claim 19 comprising apharmacologically active compound covalently attached to thegelatin-like protein.